1. Field of the Invention
The present invention relates to enzymes and more specifically to sulfotransferases that synthesize the HNK-1 carbohydrate epitope.
2. Background Information
Neural and immune cells express certain characteristic carbohydrate glycans on their cell surfaces (Jessell et al. (1985) Ann. Rev. Neurosci. 13, 227-255; Schachner and Martini (1995) Trends Neurosci. 18, 183-191; all references cited herein are incorporated by reference). One such glycan is the HNK-1 carbohydrate epitope, originally discovered by a monoclonal antibody raised against Human Natural Killer cells (Abo and Balch (1981) J. Immunol. 127, 1024-1029). The functional significance of the HNK-1 carbohydrate was first recognized as an autoantigen involved in peripheral demyelinative neuropathy. The structural analysis of glycolipids reacting with these autoantibodies led to the discovery that the HNK-1 epitope is sulfo.fwdarw.3GlcA.beta.1.fwdarw.3Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw. R (Chou et al., (1986) J. Biol. Chem. 261, 11717-11725; Ariga et al., (1987) J. Biol. Chem. 262, 848-853).
By using HNK-1-specific antibodies and carbohydrate structural studies, the HNK-1 glycan has been found in a number of neural cell adhesion molecules including N-CAM, myelin-associated glycoprotein, L1, contactin and P0 (Schachner and Martini (1995) Trends Neurosci. 18, 183-191; McGarry et al., (1983) Nature (London) 306, 376-378; Kruse et al., (1984) Nature (London) 311, 153-155; Gennarini et al., (1989) J. Neurosci. Res. 22, 1-12; Voshol et al., (1996) J. Biol. Chem. 271, 22957-22960). The studies, using either monoclonal antibodies or isolated carbohydrates, demonstrated that the HNK-1 glycan is involved in cell-cell and cell-substratum interactions (Keilhauer et al. (1985) Nature (London) 316, 728-730; Mohan et al., (1990) J. Neurochem. 54, 2024-2031).
Expression of the HNK-1 epitope is spatially and developmentally regulated, and is found on migrating neural crest cells, cerebellum and myelinating Schwann cells in motor neurons, but not on those in the sensory neurons (Bronner-Fraser (1986) Dev. Biol. 115, 44-55; Eisenman and Hawkes, (1993) J. Comp. Neurol. 335, 586-605; Martini et al., (1992) Eur. J. Neurosci. 4, 628-639). In addition, the HNK-1 carbohydrate binds to P-selectin and L-selectin (Needham and Schnaar, (1993) Proc. Natl. Acad. Sci., U.S.A. 90, 1359-1363), suggesting that interactions between immune cells and the nervous system may be mediated through binding of the HNK-1 glycan in neural cells.
The HNK-1 glycan is synthesized in a stepwise manner by adding a .beta.1,3-linked glucuronic acid to a precursor N-acetyllactosamine, followed by adding a sulfate group to GlcA.beta.1.fwdarw.3Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw.R (Jungalwala, (1994) Neurochem. Res. 19, 945-957; Chou and Jungalwala, (1993) J. Biol. Chem. 268, 330-336). A .beta.-1,3-glucuronyl transferase GlcAT-P (glycoprotein-specific glucuronyltransferase)--which forms an HNK-1 precursor carbohydrate, GlcA.beta.1.fwdarw.3Gal.beta.1.fwdarw.4GlcNAc.beta.1.fwdarw.R, in glycoproteins--has been cloned (Terayama et al., (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 6093-6098).
Desulfation experiments have shown that the sulfate group plays a critical role (Mohan et al., (1990) J. Neurochem. 54, 2024-2031). A sulfotransferase involved in the synthesis of HNK-1 has been cloned from rat cells ("raHNK-1ST") (Bakker et al., (1997) J. Biol. Chem. 272, 29942-29946.), although it is believed that the sequence of this sulfotransferase was not publicly known or available until after the date of conception or reduction to practice of the present invention. For the sake of comparison, however, the rat sequence is described herein.
Thus, HNK-1 sulfotransferases have not yet been described as of the present invention. Thus, a need exists for a HNK-1 sulfotransferase involved in synthesizing the HNK-1 glycan. The present invention satisfies this need and provides related advantages as well.